Dynamics of the multidomain fibrinolytic protein urokinase from two-dimensional NMR

Oswald, Robert E.; Bogusky, Michael J.; Bamberger, Michelle; Smith, Richard A.; Dobson, Christopher M.

doi:10.1038/337579a0

Cited by 63 publications

(53 citation statements)

References 17 publications

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“…Therefore, our data suggest that surfacebound plasminogen activators are no different to the soluble proteases in their reactivity with maspin, with neither being inhibited. The second consideration, in the case of uPA, is that our observations are consistent with the known independence of the C-terminal catalytic domain from the N-terminal uPARbinding domain (40) and our previous observations on the mechanism of enhancement of plasminogen activation by uPAR. We have shown that the catalytic activity of uPA is not affected by uPAR and that the enhanced plasminogen activation is due to the formation of catalytically favored complexes with cell-associated plasminogen (8,9,41).…”

Section: Evidence That Maspin Is Not a Protease Inhibitory Serpin 46847supporting

confidence: 71%

Maspin Inhibits Cell Migration in the Absence of Protease Inhibitory Activity

Bass

Fernández

Ellis

2002

Journal of Biological Chemistry

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Maspin is a member of the serpin family of protease inhibitors and is a tumor suppressor gene acting at the level of tumor invasion and metastasis. This in vivo activity correlates with the ability of maspin to inhibit cell migration in vitro. This behavior suggests that maspin inhibits matrix-degrading proteases, such as those of the plasminogen activation system, in a similar manner to the serpin PAI-1. However, there is controversy concerning the protease inhibitory activity of maspin. It is devoid of activity against a wide range of proteases, in common with other non-inhibitory serpins, but has recently been reported to inhibit plasminogen activators associated with cells and other biological surfaces We have compared the effects of maspin with those of PAI-1 in a range of situations in which plasminogen activation is potentiated, reflecting the biological context of this proteolytic system: urokinase-type plasminogen activator bound to its receptor on the surface of tumor cells, tissue-type plasminogen activator specifically bound to vascular smooth muscle cells, fibrin, and the prion protein. Maspin was found to have no inhibitory effect in any of these situations, in contrast to the efficient inhibition observed with PAI-1, but nevertheless maspin inhibited the migration of both tumor and vascular smooth muscle cells. We conclude that maspin is a noninhibitory serpin and that protease inhibition does not account for its activity as a tumor suppressor.

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Section: Evidence That Maspin Is Not a Protease Inhibitory Serpin 46847supporting

confidence: 71%

Maspin Inhibits Cell Migration in the Absence of Protease Inhibitory Activity

Bass

Fernández

Ellis

2002

Journal of Biological Chemistry

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“…The spectrum of the reduced peptide (Fig. 1A) contains a large number of intense intense crosspeaks observed in the spectrum are from residues with substantial independent mobility; such mobility has been observed in a number of other proteins of high molecular weight (22)(23)(24)(25). In the present case the mobile regions are likely to derive from the relatively disordered N and C termini of the Fab polypeptide chain.…”

Section: Resultsmentioning

confidence: 79%

Antigen mobility in the combining site of an anti-peptide antibody.

Cheetham

Raleigh

Griest

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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The interaction between a high-affinity antibody, raised against a peptide incorporating the loop region of hen egg lysozyme (residues 5744), and a peptide antigen corresponding to this sequence, has been probed by proton NMR. The two-dimensional correlated spectroscopy spectrum of the antibody-antigen complex shows sharp, well-resolved resonances from at least half of the bound peptide residues, indicating that the peptide retains considerable mobility when bound to the antibody. The strongly immobilized residues (which include Arg-61, do not correspond to a contiguous region in the sequence of the peptide. Examination of the crystal structure of the protein shows that these residues, although remote in sequence, are grouped together in the protein structure, forming a hydrophobic projection on the surface of the molecule. The antibody binds hen egg lysozyme with only a 10-fold lower affinity than the peptide antigen. We propose that the peptide could bind to the antibody in a conformation that brings these groups together in a manner related to that found in the native protein, accounting for the high crossreactivity.One of the most intriguing aspects of antibody recognition is the ability of some antibodies raised against peptide fragments of proteins to crossreact with intact native proteins (1-3). This phenomenon not only has considerable significance from the point of view of protein structure and folding but also has generated much interest in the possibility ofusing small peptides in a pharmacological role, particularly as synthetic vaccines (4,5). Little is known, however, about the underlying principles that determine whether a particular peptide is able to act as a good mimic of a protein epitope, primarily-because of the shortage of structural information pertaining to antibodies complexed with their peptide antigens. To our knowledge there has, for example, been only one report ofthe crystallographic structure of an anti-peptide antibody-peptide complex (6). In contrast a number of structures of anti-protein antibodies complexed with their protein antigens have been determined (7-11). As part of a long-term study in our laboratories of the structural origins of antibody recognition and protein-peptide crossreactivity, a panel of antibodies (Gloopl-GloopS) specific for the loop determinant of hen egg lysozyme was raised against a peptide immunogen that incorporated residues 57-84 of the protein sequence (12)-known as the "loop" peptide. This sequence corresponds to a large surface loop in the native protein structure and contains a disulfide bridge between residues 64 and 80. The NMR studies have focused on one of the antibodies, Gloopl. The antibody binds two forms of the loop peptide, where the disulfide is either reduced or oxidized, with equal affinity (Kd 10-8 M). The antibody is also strongly crossreactive with the native protein; it binds to hen lysozyme itself with only a 10-fold drop in affinity (12, 13). Crystallographic studies have yielded a structure for the Gloopl Fab (R.E.G. a...

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“…As the temperature is raised the domains are liberated in motion, but not chemically, assuming independent motion and they melt in an order, i.e. kringle, EGF then one of the protease domains [72]. The functional value of this differential mobility is not known.…”

Section: Gross Domain Mobility: Kringle-containing Proteinsmentioning

confidence: 99%

“…Of great interest is the power of NMR to look at proteins which are relatively large, up to 50 k Da proteins, and to isolate certain zones of interest. This needs careful temperature dependent studies and analysis of separated domains [72] as well as the use of a great variety of pulse sequences [15] and of nuclei other than protons.9. In this article 1 have illustrated the different possibilities using work in my own group.…”

mentioning

confidence: 99%

NMR studies of mobility within protein structure

Williams

1989

European Journal of Biochemistry

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NMR studies of dynamics within structure have revealed that a quite new approach to protein structure and its relation to function is necessary. This approach requires the consideration in detail of the following :1. Local movements of groups and small segments to allow fast recognition and fitting. The motion concerns on/off rates as well as binding. The observations affect surface/surface recognition, e.g. of antigen/antibody as well as of substrate and protein.2. Somewhat larger interdomain or N-and C-terminal segments which allow rearrangement. Cases in point are the movement of segments in blood-clotting proteins or in histones.3. Relative motion of helices in hinges. These actions are likely in such enzymes as kinases and P-450 cytochromes.4. Relative motion of helices within domains (relative to other helices or sheets) in mechanical devices (triggers) e.g. in cdlmodulin.5. General motion in random proteins. Examples extend from rubber-like proteins (entropy sensors), some glycoproteins, to proteins carrying peptide hormones to be generated only after hydrolysis.6. Order -+ disorder transitions locally as in osteocalcin and metallothionine. 7. Swinging arm motions associated with special sequences such as (Ala-Pro),. 8. Of great interest is the power of NMR to look at proteins which are relatively large, up to 50 k Da proteins, and to isolate certain zones of interest. This needs careful temperature dependent studies and analysis of separated domains [72] as well as the use of a great variety of pulse sequences [15] and of nuclei other than protons.9. In this article 1 have illustrated the different possibilities using work in my own group. This is done to lessen the burden of extensive review. I fully realise that the range of examples is now large. I would stress though that the production of the necessary technology was the endeavour of several of us within the Oxford Enzyme Group from 1970 to 1985, i.e. from 270-600 MHz Fourier-transform NMR spectroscopy.10. While all of these features have been demonstrated by NMR methods there are parallel developments both using X-ray diffraction methods and theoretical approaches. All these procedures are changing the view of protein structure to one which incorporates dynamics all the way from conventional vibronic/rotational coupling to the disordered motions characteristic of random polymers. It is the understanding of dynamics that leads to an appreciation of function.Correspondence ta R. J. P. Williams, Inorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, EnglandAbbreviations. FUR protein, iron-uptake regulatory protein; NOE, nuclear Overhauser effect; COSY, correlated spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; Gla, y-carboxyglutamate.No&. While this article was being completed, a review was published in the European Journal of Biochemistry [76] which has summarised very effectively some of the problems faced by all users of NMR methods who attempt to describe either structure or dynamics of large flexible...

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Dynamics of the multidomain fibrinolytic protein urokinase from two-dimensional NMR

Cited by 63 publications

References 17 publications

Maspin Inhibits Cell Migration in the Absence of Protease Inhibitory Activity

Maspin Inhibits Cell Migration in the Absence of Protease Inhibitory Activity

Antigen mobility in the combining site of an anti-peptide antibody.

NMR studies of mobility within protein structure

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